The classical fluorescence microscopy is using the capability of certain substances to emit light with a longer wavelength (lower energy) although excited by a shorter wavelength (normally UV-wavelengths). In this case the energy of the absorbed photons is transfered to excite electrons in a state E2. Then the electrons loose part of their energy, because of radiationless transitions, and accumulate in a long lasting state at the lower boundry of the excited state. At the transition into the ground state E1photons are emitted which are observed as fluorescence. The lifetime of the excited state defines the fluorescence decay curve. If one photon doesn't suffice the necessary energy to excite the electron, it is possible that multiple photons are absorbed simultaniously. The probability of a multiphoton process is highly dependent on the photon density and therefore on the intensity of the radiation. With ultrashort laserpulses multiphoton absorption in fluorescent molecules can be observed at picojoule-energies. In this case the emitted fluorescence has a shorter wavelength (contrary to the one-photon process) than the absorbed photons. That is why fluorescence can also be obtained with NIR-Lasers and the more dangerous or at least contentious UV radiation can be avoided. The multiphoton-excitation has another advantage for three-dimensional imaging compared to the classical one-photon fluorescence. Due to the strong dependancy on the intensity, fluorescence is not excited in the complete region of radiation, but in a small space around the focus area. This results in a high spatial resolution even lower than the diffraction minimum and a depth resolution normally achieved in classical confocal scanning-microscopes only with a pinhole.
Second harmonic generation (SHG; also called frequency doubling) is a nonlinear optical process, in which photons interact with a nonlinear material. They are effectively combined to form new photons with twice the energy, and therefore twice the frequency and half the wavelength of the initial photons. In recent years, SHG has been extended to biological applications. Researchers Leslie Loew and Paul Campagnola at the University of Connecticut have applied SHG to imaging of molecules that are intrinsically second-harmonic-active in live cells, such as collagen, while Joshua Salafsky is pioneering the technique's use for studying biological molecules by labelling them with second-harmonic-active tags, in particular as a means to detect conformational change at any site and in real time. SHG-active unnatural amino acids can also be used as probes.